Project summary: We have identified old intragraft DC as drivers of an IL-17 driven immune response in recipient animals. Mechanisms that activate old DCs, however, remain yet to be determined. Our new data found a systemic increase of mitochondrial DNA (mt-DNA) in aging and a compromised clearance of senescent cells that was responsible for the augmented immunogenicity of old DCs and the subsequent Th17 driven alloimmune responses. In specific aim 1 we will restore senescent cell clearance in old animal and assess DCs immunogenicity in models of IRI and transplantation. We have also been able to publish that old T cells have an impaired IFN? production, IL-2 receptor activation and altered T/DCs communication. Moreover, we published that Tacrolimus (TAC) a mainstay clinical immunosuppressant works in an age-specific way. Mechanisms involved included an age-specific inhibition of IL-2 production and a compromised proliferation of old T-cells. In addition, intracellular calcineurin levels and Ca2+ influx, two critical pathways in T cell activation were suppressed in an age-specific way. We have now further novel preliminary data providing the opportunity to study relevant mechanisms of age-specific alloimmunity in-depth: In a sensitization model, we observed that old sensitized animals reject secondary third party grafts in an accelerated fashion. This process was not observed in young animals, indicating that aging may promote unspecific innate immunity to the detriment of adaptive immune responses. We also observed a downregulation of key genes of autophagy in old CD4+ T- cells, while key players of innate immunity such as monocytes showed an increased expression of autophagy. In specific aim 2, we will therefore elucidate the role of an age-dependent autophagy, testing a shift in the balance of innate and adaptive immunity in aging. We also want to understand the mechanisms behind age- specific immunosuppression. Our preliminary data show that old CD4+ T cells have a compromised switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, a hallmark of T cell activation. Furthermore, old CD4+ T cells that remained on OXPHOS demonstrated significantly limited expression of pro-inflammatory cytokines and an upregulation of PD1, a major marker for T cell exhaustion. In our third aim, we will therefore dissect the effects of an age-dependent imbalance of aerobic glycolysis and OXPHOS and will examine how immunosuppressants affect T-cell metabolism in an age-specific fashion. Our overall hypothesis is that aging is associated with increased amounts of circulating cell free mt-DNA that augments immunogenicity, while, on the recipient side, changes in autophagy and metabolic profiles shape age-specific alloimmune responses and the effects of immunosuppressants. We believe that our proposal addresses a significant clinical issue. With the comprehensive experiments proposed, we expect to contribute substantially to improved understanding of effects of aging on alloimmunity and transplantation outcomes.